Identification of stabilizers of multimeric proteins

ABSTRACT

Disclosed herein are compounds and compositions thereof which find use in increasing stability of TTR tetramers reducing its tendency to misfold and form aggregates. Also provided herein are methods for using these compounds and compositions for increasing stability of TTR and thereby decreasing aggegate formation by TTR. Also disclosed herein are methods to screen for candidate compounds that increase stability of TTR. Also disclosed herein are heterobifunctional compounds that include a TTR binding compound connected to a targeting moiety via a linker, for use in disrupting PPis of a target protein.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/092,446, filed on Apr. 6, 2016, now U.S. Pat. No. 10,039,726, whichis a continuation of U.S. patent application Ser. No. 14/531,888, filedNov. 3, 2014, now U.S. Pat. No. 9,308,209, which is a continuation ofU.S. patent Ser. No. 13/696,505, now U.S. Pat. No. 8,877,795, which is a371 of PCT/US2011/035350, filed May 5, 2011, which claims the benefit ofU.S. Provisional Patent Application No. 61/332,638, filed May 7, 2010,which applications are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. PN2EY016525 awarded by National Institute of Health. The government hascertain rights in this invention.

INTRODUCTION

Targeting protein protein interactions (PPIs) is of therapeuticinterest. To date approved inhibitors of PPIs are proteins rather thansmall-molecule inhibitors. For example, therapeutic monoclonalantibodies (mAbs) are used in treating cancer, autoimmune, infectiousand neurodegenerative diseases. Therapeutic mAbs are costly tomanufacture, they require administration by injection and can illicit animmune-response in the patient. For these reasons the development ofsmall-molecule inhibitors of PPIs is of interest.

The cytokine tumor-necrosis factor alpha (TNF-α) plays an important rolein the inflammatory response to tissue injury and various viral andbacterial infections. TNF-α forms homotrimers, which bind to the TNF-αreceptors 1 and 2 and induce receptor trimerization. Depending on thecellular context, trimerization of the TNF receptor 1 (TNFR1) can leadto activation of the immune system by the NFκB signaling pathway. Sinceaberrantly increased TNF-α activity may also lead to tissue damage,inhibitors of TNF-α are of clinical interest for the treatment ofautoimmune diseases, such as rheumatoid arthritis or Crohn's disease.These pathological conditions are currently being treated withanti-TNF-α antibodies and soluble receptor molecules, which act bysequestering TNF-α. Neutralising anti-TNF-α antibodies and soluble TNFreceptor preparations have anti-inflammatory activities in clinicalstudies, particularly in rheumatoid arthritis. TNF-α also activatesosteoclasts both by itself and in synergy with RANKL and is a target forthe treatment of bone disorders such as osteoporosis. Inhibition of theinteraction between TNF-α and its receptor TNFR1 usinghetero-bifunctional molecules is of interest for inhibition of TNF-αactivity.

Interleukin-2 (IL-2) is a 15.5 kD cytokine that has a predominant rolein the growth of activated T cells. IL-2 stimulates T-cell proliferationby binding on the T-cell surface with picomolar affinity to aheterotrimeric receptor complex consisting of α, β, and γ chains. TheIL2/IL-2Rα interaction is a target for therapeutic modulation becausethe IL2Rα is not expressed on resting T and B cells but is continuouslyexpressed by the abnormal T cells of patients with forms of leukemia,autoimmunity, and organ transplant rejection. Antibodies that recognizethe a receptor subunit (IL-2Rα) and block IL-2 binding are clinicallyeffective as immunosuppressive agents. Inhibition of the interactionbetween IL-2 and IL-2Ra using hetero-bifunctional molecules is ofinterest for inhibition of IL-2 activity.

The deposition of a normally soluble protein into amyloid fibrils is ahallmark of human amyloid diseases. Conformational changes aresufficient for the conversion of a number of normally soluble humanproteins into amyloid fibrils, including the immunoglobulin lightchains, lysozyme, and transthyretin (TTR), and variants thereof. Fibrilformation is believed to be intimately involved in the pathologicalmechanism of human amyloid disease based on the demonstratedneurotoxicity of amyloid fibrils produced in vitro, the observation oflower levels of amyloid in age-matched controls relative to Alzheimerdisease patients, and the correlation of improved health with theclearance of amyloid in Familial Amyloid polyneuropathy (FAP) patients,where liver transplantation is used to replace mutant TTR with wild-typeTTR.

There is an interest in identifying ways to prevent the conformationalchanges that result in the formation of amyloid fibrils. In the case ofTTR, it has been determined that stabilizing TTR in its tetrameric forminhibits the formation of TTR amyloids.

Transthyretin (TTR or prealbumin) is a 55 kDa homotetrameric proteinpresent in blood and cerebrospinal fluid. TTR is primarily synthesizedin the liver. TTR transports holoretinol binding protein (RBP) andL-thyroxine (T4) in the blood and cerebrospinal fluid. The misfolding ofwild type TTR (WT-TTR) or one of >100 different mutated variants,through dissociation to non-native monomeric intermediates thataggregate and polymerize into amyloid fibrils, is associated withvarious TTR amyloid diseases.

Several classes of small molecules have been reported to inhibit TTRamyloid formation by binding to the T4-binding sites in TTR andkinetically stabilizing its quaternary structure. TTR has two identicalfunnel-shaped T4-binding sites located at its dimer-dimer interface. TTRis abundant in plasma (1.8-5.4 μM tetramer concentration) and smallmolecule binders of TTR should not compete with the natural ligand (T4)as <1% of TTR in the plasma is typically bound to T4. Recently,Tafamidis, a TTR kinetic stabilizer, completed a successful phase II/IIIclinical trials for FAP. The nonsteroidal anti-inflammatory drug (NSAID)diflunisal also showed promising results in clinical trial for FAP.

Most of the other TTR stabilizers reported are structurally based ontypical biaryl and halogenated biaryl analogues of T4 and NSAID-likecompounds, respectively.

There is a need for discovery of TTR kinetic stabilizers.

SUMMARY

Disclosed herein are compounds and compositions thereof which find usein increasing stability of proteins particularly proteins that tend tomisfold and form aggregates. Also provided herein are methods for usingthese compounds and compositions for increasing stability of proteinsand thereby decreasing aggregate formation by these proteins. Alsodisclosed herein are methods to screen for candidate compounds thatincrease stability of proteins. Also disclosed herein areheterobifunctional compounds that include a TTR binding compoundconnected to a targeting moiety via a linker, for use in disrupting PPIsof a target protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A depicts the structure of compounds 1-5. FIGS. 1B-1E depictresults of calorimetric titration of ligands against TTR. (FIG. 1B)K_(d) for 1=72.5±4.7 nM. (FIG. 1C) K_(d) for 2>3289 nM nM. (FIG. 1D)K_(d) for 4=284.9±58.1 nM. (FIG. 1E) K_(d) for diclofenac=370.4±145.4nM). Raw data (top) and integrated heats (bottom) from the titration ofTTR (2 μM) with ligands (25 μM). The solid red line through theintegrated heats represents the best fit binding isotherm to aone-to-one binding model.

FIGS. 2A-2C depict results from the assessment of the binding affinityof probe 5 to TTR. (FIG. 2A) calorimetric titration of 5 against TTR(K_(d)=819.7±129.7 nM). Raw data (top) and integrated heats (bottom)from the titration of TTR (2 μM) with probe 5 (25 μM). The solid redline through the integrated heats represents the best fit bindingisotherm to a one-to-one binding model. (FIG. 2B) Fluorescencepolarization saturation binding between 5 (100 nM) and increasingconcentration of TTR (5 nM to 10 μM) (FIG. 2C) Displacement of 5 fromTTR by increasing concentration (10 nM-50 μM) of ligand 1 (K_(app)=0.231μM, R²=0.997). FP Assays were performed in triplicate and the error barsrepresent STDV.

FIGS. 3A-3C depict displacement FP assay of TTR ligands. (FIG. 3A) 2(K_(app)>50 μM). (FIG. 3B) Thyroxine T4 (K_(app)=0.186 μM, R²=0.998).(FIG. 3C) Diclofenac (K_(app)=4.66 μM, R²=0.999).

FIG. 4A shows average FP mP values for probe 5 incubated with TTR (MAX,♦) or buffer (MIN, ●). FIG. 4B shows Z′ factor values calculated foreach plate.

FIGS. 5A and 5B show evaluation of the potency of HTS ligands as TTRaggregation inhibitors. (FIG. 5A) Percentage of TTR (3.6 μM) fibrilformation in the presence of ligands (7.2 μM) relative to aggregation inthe absence of ligands (denoted 100%) at 72 hours. (FIG. 5B) Comparisonof TTR (3.6 μM) aggregation inhibition in the presence ofsubstoichiometric amounts of ligands (3.0 μM) relative to diclofenac.

FIGS. 6A and 6B show results from measurement of cytotoxicity of TTRligands (8 μM) on human AC16 cardiac cells (FIG. 6A) and human IMR-32neuroblastoma cells (FIG. 6B). FIGS. 6C and 6D illustrate inhibition ofV122 TTR and WT TTR cytotoxicity in human AC16 cardiac cells and IMR-32neuroblastoma cells. (FIG. 6C) V1221 TTR was pre-incubated in theabsence (V1221 TTR) or presence of ligands for 24 h at 4° C. and thenadded to human AC16 cardiac cell culture. (FIG. 6D) WT TTR pre-incubatedin the absence (WT TTR) or presence of ligands for 24 h at 4° C. andthen added to human IM-32 neuroblastoma cell culture.

FIGS. 7A-7D illustrate calorimetric titration of Ro 41-0960(K_(d)=184.5±26.5 nM) (FIG. 7A); niflumic acid (K_(d)=591.7±146.6 nM)(FIG. 7B); compound 7 (K_(d)=320.5±27.6 nM) (FIG. 7C); and compound 14(K_(d)=245.1±40.1 nM) (FIG. 7D) for binding to TTR. Raw data (top) andintegrated heats (bottom) from the titration of TTR (2 μM) with ligand(25 μM). The solid red line through the integrated heats represents thebest fit binding isotherm to a one-to-one binding model. FIG. 7E shows aSPR sensogram showing concentration-dependent binding of ligand 7 towild type biotinylated TTR on a streptavidin-coupled surface over aconcentration of 1 nM to 2.2 in order of increasing RUs. Normalized RUsare plotted over a time course. Equilibrium binding analysis (inset)indicates a K_(d) of 57.91±13.2 nM (SD).

FIGS. 8A-8C depict assessment of the binding TTR ligands using SPR.(FIG. 8A) SPR sensogram showing concentration-dependent binding ofligand Ro-41-0960 to wild type biotinylated TTR on astreptavidin-coupled surface over a concentration of 90 nM to 3 μM, inorder of increasing RUs. Normalized RUs are plotted over a time course.(FIG. 8B) SPR sensogram for niflumic acid binding to TTR. Equilibriumbinding analysis indicates a K_(d) of 186.1±23.8 nM(k_(on)=2.81×10⁶±3.6×10⁵ M⁻¹ s⁻¹ and k_(off)=0.523±3.6×10⁻⁶ s⁻¹) (SD).(FIG. 8C) SPR sensogram for diclofenac binding to TTR. Equilibriumbinding analysis indicates a K_(d) of 123.5±8.91 nM(k_(on)=8.18×10⁶±5.9×10⁵ M⁻¹ s⁻¹ and k_(off)=1.01±0.003 s⁻¹) (SD).

FIG. 9 depicts the selection of the linker of a subjectheterobifunctional compound using computational chemistry. R and P_(R)are a recruiting moiety and recruited protein respectively. T and P_(T)are a targeting moiety and target protein, respectively.

FIG. 10 depicts a heterobifunctional molecule (top) and the chemicalstructures of exemplary TTR ligands, linkers and targeting moieties, theIL2 binder Ro26-4550 (IC50 3-6 μM) and the TNFα ligand (IC50=22 μM)(bottom). The arrows show exemplary points of attachment of the linkerto the targeting moiety.

FIG. 11A-B shows SPR (Biacore) and ITC data and TTR binding affinitiesof two exemplary compounds identified using the FP-based HTS assay.

FIG. 12 illustrates the cell viability of primary cardiomyocytes assayed48 hours after addition of TTR binders (10 μM).

DEFINITIONS

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature or during chemical synthesis. Isolatedcompounds are usually at least about 80% pure, or at least about 90%pure, at least about 98% pure, or at least about 99% pure, by weight.The present invention is meant to encompass diastereomers as well astheir racemic and resolved, enantiomerically pure forms andpharmaceutically acceptable salts thereof.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease, (2) inhibiting the disease, i.e.,arresting or reducing the development of the disease or its symptoms, or(3) relieving the disease, i.e., causing regression of the disease orits clinical symptoms.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

The terms “subject” and “patient” mean a mammal that may have a need forthe pharmaceutical methods, compositions and treatments describedherein. Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), and other subjects. Humans and non-human animalshaving commercial importance (e.g., livestock and domesticated animals)are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes both one andmore than one such excipient, diluent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” ispreferably sterile, and free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal and the like.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

A “pharmaceutically acceptable solvate or hydrate” of a compound of theinvention means a solvate or hydrate complex that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound, and includes, but is not limited to, complexes of acompound of the invention with one or more solvent or water molecules,or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solventor water molecules.

The term “organic group” and “organic radical” as used herein means anycarbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combination thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

“Organic groups” may be functionalized or otherwise comprise additionalfunctionalities associated with the organic group, such as carboxyl,amino, hydroxyl, and the like, which may be protected or unprotected.For example, the phrase “alkyl group” is intended to include not onlypure open chain saturated hydrocarbon alkyl substituents, such asmethyl, ethyl, propyl, t-butyl, and the like, but also alkylsubstituents bearing further substituents known in the art, such ashydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino,carboxyl, etc. Thus, “alkyl group” includes ethers, esters, haloalkyls,nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent. Halogens of particular interest include chloro and bromogroups.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to C₄ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables (e.g.,-J, ═W—, —X═, ═Y—, —Z═, -Q, —R^(V1), —R^(V2), —R^(V3), —R^(V4), —R^(T),—R^(TT), -Q^(CA), -Q^(HA), —R^(PP), —R^(R), —R^(RA), -L^(R), -M^(R),—R^(R), —R^(RR), —R^(J), -M^(J), —R^(N), —R^(J1), —R^(J2), —R^(J3),—R^(J4), —R^(J5), —R^(J6), -L^(J)-, —R^(J2XX), —R^(JJ), —R^(JJJ),L^(JJJ)-, —R^(P2), —R^(P3), —R^(P4), —R^(P5), —R^(P6), —R^(P2R),—R^(P2RA), —R^(P3R), —R^(P3RA), —R^(P4R), —R^(P5R), —R^(P5RA), —R^(P6R),—R^(P6RA), —R^(AK), etc.) are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed, to the extent that suchcombinations embrace compounds that are stable compounds (i.e.,compounds that can be isolated, characterized, and tested for biologicalactivity). In addition, all sub-combinations of the chemical groupslisted in the embodiments describing such variables are alsospecifically embraced by the present invention and are disclosed hereinjust as if each and every such sub-combination of chemical groups wasindividually and explicitly disclosed herein

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Overview

The present disclosure is based on the identification of compounds thatbind to a TTR tetramer in the presence of a TTR ligand known to bind andstabilize TTR tetramer. These compounds stabilize TTR tetramers reducingthe formation of TTR amyloid fibrils. These compounds find use in thepreparation of heterobifunctional compounds that recruit TTR for use indisrupting PPIs.

The present disclosure also provides a method of identifying a ligandfor TTR. Generally, the method uses a fluorescence polarization(FP)-probe comprising a ligand for TTR and a fluorescent moiety attachedto the ligand by a linker. The FP probe binds to TTR to form a TTR-FPprobe complex. The method includes: a) contacting a TTR-FP probe complexwith a candidate compound, wherein the TTR-FP probe complex generates aFP signal; and b) determining the FP signal, wherein a decrease in theFP signal indicates the candidate compound binds to TTR and is a ligandfor TTR.

Compositions

Provided herein are compounds that may be used to stabilize TTRtetramers reducing TTR amyloid fibril formation. These compounds can beincorporated into a variety of formulations for therapeuticadministration by a variety of routes. More particularly, the compoundsdisclosed herein can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers,diluents, excipients and/or adjuvants. In most embodiments, theformulations are free of detectable DMSO (dimethyl sulfoxide), which isnot a pharmaceutically acceptable carrier, diluent, excipient, oradjuvant.

Compounds

In some embodiments, a compound of the invention is of the structure ofFormula I:

where R¹ is an aryl or a hetereocyclic group; and

Z¹, Z² and Z³ are independently O, S, NH or NR², where R² is hydrogen,an alkyl or an aryl.

In some embodiments, a compound is of the structure of Formula I where:

R¹ is a phenyl, or a five-membered heterocyclic group;

Z¹ is NH;

Z² is NH or O; and

Z³ is S or NR³, where R³ is lower alkyl.

In some embodiments, a compound of the invention is of the structure ofFormula II:

where Z¹ is NH;

Z² is NH or O;

Z³ is S or NR³, where R³ is lower alkyl; and

R⁴ is one or more groups, each R⁴ independently selected from hydrogen,an alkyl, an aryl, an alkoxy, an aryloxy, an acetyl, a carboxy, aformyl, an amido, a hydroxyl, a heterocyclic group, a halo, a nitro anda cyano, where optionally two or more R⁴ groups may be cyclicallylinked. In some embodiments, in Formula II, Z¹ and Z² are NH and Z³ isNCH₃.

In some embodiments, in the structure of Formula II at least one R⁴group is selected from a carboxy, a formyl and a hydroxy, and isattached at the 2-position of the phenyl ring. In some embodiments, inthe structure of Formula II at least one R⁴ group is selected from acarboxy, a formyl and a hydroxy, and is attached at the 3-position ofthe phenyl ring. In some embodiments, in the structure of Formula II atleast one R⁴ group is selected from a carboxy, a formyl and a hydroxyand is attached at the 4-position of the phenyl ring.

In some embodiments, a compound of the invention is of the structure ofFormula III:

where R⁵ is one or more groups, each R⁵ independently selected fromhydrogen, an alkyl, an aryl, an alkoxy, an aryloxy, an acetyl, acarboxy, a formyl, an amido, a hydroxyl, a heterocyclic group, a halo, anitro and a cyano, where optionally two or more R⁵ groups may becyclically linked. In some embodiments, in structure of Formula III, twoR⁵ groups on adjacent carbons of the ring may be cyclically linked toform a fused phenyl ring.

In some embodiments, a compound of the invention is of the structure ofFormula III where:

R⁵ is one or more groups, each R⁵ independently selected from abenzyloxy group, a trifluoromethyl, a halo, a carboxy, a formyl, a loweralkyl, a hydroxyl, a lower alkoxy and a phenyl.

In some embodiments, in the structure of Formula III at least one R⁵group is selected from a carboxy, a formyl and a hydroxy, and isattached at the 2-position of the phenyl ring. In some embodiments, inthe structure of Formula III at least one R⁵ group is selected from acarboxy, a formyl and a hydroxy, and is attached at the 3-position ofthe phenyl ring. In some embodiments, in the structure of Formula III atleast one R⁵ group is selected from a carboxy, a formyl and a hydroxyand is attached at the 4-position of the phenyl ring.

In some embodiments, a compound of the invention is of the structure ofFormula IV:

where R⁶ is selected from hydrogen, an alkyl, an aryl, an alkoxy, anaryloxy, an acetyl, a carboxy, a formyl, an amido, a hydroxyl, aheterocyclic group, a halo, a nitro and a cyano.

In some embodiments, a compound of the invention is of the structure ofFormula IV where R⁶ is selected from a benzyloxy group, atrifluoromethyl, a bromo, a chloro, a methyl, a hydroxyl, a methoxy anda phenyl.

In some embodiments, a compound of the invention is of the structure ofone of Formulas V, VI or VII:

where R⁸, R¹⁰ and R¹³ are independently one or more groups, each R⁸, R¹⁰and R¹³ independently selected from hydrogen, an alkyl, an aryl, analkoxy, an aryloxy, an acetyl, a carboxy, a formyl, an amido, ahydroxyl, a heterocyclic group, a halo, a nitro and a cyano, whereoptionally two or more R⁸, R¹⁰ or R¹³ groups may be cyclically linked;and

R⁷, R⁹, R¹¹ and R¹² are independently selected from hydrogen, an alkyl,an aryl, an acetyl, a carboxy, a formyl, an amido, a sulfonyl, asulfinyl, a thio, an acetyl and an amino.

In some embodiments, a compound of the invention is of the structure ofone of Formulas V, VI or VII where R⁸, R¹⁰ and R¹³ are independently oneor more groups, each R⁸, R¹⁰ and R¹³ independently selected from abenzyloxy group, a trifluoromethyl, a halo, a carboxy, a formyl, amethyl, a hydroxyl, a methoxy and a phenyl; and

R⁷, R⁹, R¹¹ and R¹² are independently selected from methylsulfinyl,methylsulfonyl, a lower alkyl thioether, SH and a lower alkyl. In someembodiments, in Formula V, R⁷ is —SCH₃ or —SO₂CH₃. In some embodiments,in Formula VI, R⁹ is —SCH₃ or —SO₇CH₃.

In some embodiments, in the structure of one of Formulas V, VI and VIIat least one R⁸, R¹⁰ or R¹³ group is selected from a carboxy, a formyland a hydroxy, and is attached at the 2-position of the phenyl ring. Insome embodiments, in the structure of Formulas V, VI and VII at leastone R⁸, R¹⁰ or R¹³ group is selected from a carboxy, a formyl and ahydroxy, and is attached at the 3-position of the phenyl ring. In someembodiments, in the structure of Formulas V, VI and VII at least one R⁸,R¹⁰ or R¹³ group is selected from a carboxy, a formyl and a hydroxy andis attached at the 4-position of the phenyl ring.

In some embodiments, a compound of the invention is of the structure ofFormula VIII:

where Z⁴ is a single bond, a methylene, an aminomethylene, ahydroxymethylene or a linker of about 1 to 3 atoms in length; and

R¹⁴ and R¹⁵ are independently one or more groups, each R¹⁴ and R¹⁵ groupindependently selected from hydrogen, an alkyl, an aryl, an alkoxy, anaryloxy, an acetyl, a carboxy, a formyl, an amido, a hydroxyl, aheterocyclic group, a halo, a nitro and a cyano, where optionally two ormore R¹⁴ or R¹⁵ groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofone of Formulas IX, X or XI:

where R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are independently one or moregroups, each R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ group independentlyselected from hydrogen, an alkyl, an aryl, an alkoxy, an aryloxy, anacetyl, a carboxy, a formyl, an amido, a hydroxyl, a heterocyclic group,a halo, a nitro and a cyano, where optionally two or more R¹⁶, R¹⁷, R¹⁸,R¹⁹, R²⁰ or R²¹ groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofone of Formulas IX, X or XI where R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ areindependently one or more groups, each R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹group independently selected from a lower alkoxy, a trifluoromethyl, acarboxy, a formyl, a lower alkyl, a hydroxyl, a nitro and a halo.

In some embodiments, in the structure of one of Formulas VIII, IX, X andXI at least one R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ or R²¹ group isselected from a carboxy, a formyl and a hydroxy, and is attached at the2-position of the phenyl ring. In some embodiments, in the structure ofone of Formulas VIII, IX, X and XI at least one R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸,R¹⁹, R²⁰ or R²¹ group is selected from a carboxy, a formyl and ahydroxy, and is attached at the 3-position of the phenyl ring. In someembodiments, in the structure of Formulas VIII, IX, X and XI at leastone R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰ or R²¹ group is selected from acarboxy, a formyl and a hydroxy and is attached at the 4-position of thephenyl ring.

In some embodiments, a compound of the invention is of the structure ofFormula XII:

where R²⁵ is independently selected from hydrogen, an alkyl, aheterocyclic group, an aryl, a thio, cyano, an alkoxy, an aryloxy, ahalo, and a hydroxyl; and

R²⁷ and R²⁸ are independently one or more groups, each R²⁷ and R²⁸independently selected from hydrogen, an alkyl, an aryl, an alkoxy, anaryloxy, an acetyl, a carboxy, a formyl, an amido, a hydroxyl, aheterocyclic group, a halo, a nitro and a cyano, where optionally two ormore R²⁷ or R²⁸ groups may be cyclically linked.

In some embodiments, in the structure of Formula XII at least one R²⁷ orR²⁸ group is selected from a carboxy, a formyl and a hydroxy, and isattached at the 2-position of the phenyl ring. In some embodiments, inthe structure of one of Formula XII at least one R²⁷ or R²⁸ group isselected from a carboxy, a formyl and a hydroxy, and is attached at the3-position of the phenyl ring. In some embodiments, in the structure ofFormula XII at least one R²⁷ or R²⁸ group is selected from a carboxy, aformyl and a hydroxy and is attached at the 4-position of the phenylring.

In some embodiments, a compound of the invention is of the structure ofFormula XIII:

where R³⁰ and R³¹ are independently selected from a hydrogen, an alkyl,a heterocyclic group, an aryl, a thio, cyano, an alkoxy, an aryloxy, ahalo and a hydroxyl;

R³² and R³³ are independently selected from hydrogen, an alkyl, aheterocyclic group and an aryl; and

R³⁴ is selected from hydrogen, an alkyl, a heterocyclic group and anaryl, where optionally R³⁴ and R³² may be cyclically linked to form afused 6-membered ring.

In some embodiments, a compound is of the structure of Formula XIIIwhere R³⁰ and R³¹ are independently selected from cyano and benzylthio;R³² and R³³ are independently selected from a hydrogen, an alkyl, aheterocyclic group and an aryl; and R³⁴ is hydrogen.

In some embodiments, a compound of the invention is of the structure ofFormula XIV:

where R³⁵, R³⁶, R³⁷, R³⁸ and R³⁹ are independently selected fromhydrogen, an alkyl, an aryl, an acetyl, a carboxy, a formyl, an amido, aheterocyclic group, a thio, cyano, a nitro, an alkoxy, an aryloxy, ahalo and a hydroxyl.

In some embodiments, a compound of the invention is of the structure ofFormula XIV where one of R³⁸ and R³⁹ is CO—R⁴⁰ where R⁴⁰ is selectedfrom hydrogen, hydroxyl, an alkyl, an aryl, an amino and an alkoxy; andthe other of R³⁸ and R³⁹ is selected from a hydrogen, an alkyl, an aryl,a heterocyclic group, a thio, cyano, a nitro, an alkoxy, an aryloxy, ahalo and a hydroxyl.

In some embodiments, a compound of the invention is of the structure ofone of Formulas XV and XVI:

where R⁴¹ and R⁴² are as defined above for R³⁵ and R³⁶; and R⁴³ ishydrogen, an amino or hydroxyl.

In some embodiments, a compound of the invention is of the structure ofFormula XVII:

where L is a linker of about 1 to 8 atoms in length;

R⁵¹ and R⁵² are selected from hydrogen, an alkyl, an aryl, an alkoxy, anaryloxy, a hydroxyl, a heterocyclic group, a halo, a nitro and a cyano;

R⁵³ is selected from hydrogen, an alkyl, an aryl and a heterocyclicgroup; and

R⁵⁴ is selected from an aryl and a heterocyclic group.

In some embodiments, a compound is of the structure of Formula XVIII:

where n is 1, 2, 3 or 4;

R⁵⁵, R⁵⁶, and R⁵⁷ are as defined above for R⁵¹, R⁵², and R⁵³; and

R⁵⁸ is selected from an aryl, an aryloxy and a heterocyclic group. Insome embodiments, R⁵⁸ has the structure:

In some embodiments, a compound of the invention is of the structure ofFormula XVIII where n is 2 or 3; R⁵⁵ and R⁵⁶ are methyl; R⁵⁷ ishydrogen; and R⁵⁸ is selected from an aryl, an aryloxy and aheterocyclic group.

In some embodiments, a compound of the invention is of the structure ofone of Formulas XIX and XX:

where L is a linker of about 1 to 8 atoms in length;

n is 1, 2, 3 or 4;

R⁶¹, R⁶², and R⁶³ are as defined above for R⁵¹, R⁵², and R⁵³; and

R⁶⁴ and R⁶⁸ are independently one or more groups, each R⁶⁴ and R⁶⁸independently selected from an alkyl, an aryl, an alkoxy, an aryloxy, anacetyl, a carboxy, a formyl, an amido, a hydroxyl, a heterocyclic group,a halo, a nitro and a cyano, where optionally two or more R⁶⁴ or R⁶⁸groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofone of Formulas XIX and XX where n is 2 or 3; R⁶¹, R⁶², R⁶⁵ and R⁶⁶ aremethyl; R⁶³ is R⁶⁷ are hydrogen; and R⁶⁴ and R⁶⁷ are independentlyselected from an aryl, an aryloxy and a heterocyclic group.

In some embodiments, in the structure of one of Formulas XIX and XX, atleast one R⁶⁴ or R⁶⁸ group is selected from a carboxy, a formyl and ahydroxy, and is attached at the 2-position of the phenyl ring. In someembodiments, in the structure of one of Formulas XIX and XX, at leastone R⁶⁴ or R⁶⁸ group is selected from a carboxy, a formyl and a hydroxy,and is attached at the 3-position of the phenyl ring. In someembodiments, in the structure of one of Formulas XIX and XX, at leastone R⁶⁴ or R⁶⁸ group is selected from a carboxy, a formyl and a hydroxyand is attached at the 4-position of the phenyl ring.

In some embodiments, a compound of the invention is of the structure ofFormula XXI:

where Z⁵ is a 5-membered heterocycle, a keto, a ketomethylene, ahydroxymethylene, a sulfonylamino or a amidomethylene; and

R⁷¹ and R⁷² are independently one or more groups, each R⁷¹ and R⁷²independently selected from an alkyl, an aryl, an alkoxy, an aryloxy, anacetyl, a carboxy, a formyl, an amido, a hydroxyl, a heterocyclic group,a halo, a nitro and a cyano, where optionally two or more R⁷¹ or R⁷²groups may be cyclically linked.

In some embodiments, in the structure of Formula XXI at least one R⁷¹ orR⁷² group is selected from a carboxy, a formyl and a hydroxy, and isattached at the 2-position of the phenyl ring. In some embodiments, inthe structure of Formula XXI at least one R⁷¹ or R⁷² group is selectedfrom a carboxy, a formyl and a hydroxy, and is attached at the3-position of the phenyl ring. In some embodiments, in the structure ofFormula XXI at least one R⁷¹ or R⁷² group is selected from a carboxy, aformyl and a hydroxy and is attached at the 4-position of the phenylring.

In some embodiments, a compound of the invention is of the structure ofFormula XXII:

where R⁷³ and R⁷⁴ are independently one or more groups, each R⁷³ and R⁷⁴independently selected from an alkyl, an aryl, an alkoxy, an aryloxy, anacetyl, a carboxy, a formyl, an amido, a hydroxyl, a heterocyclic group,a halo, a nitro and a cyano, where optionally two or more R⁷³ or R⁷⁴groups may be cyclically linked.

In some embodiments, in the structure of Formula XXII at least one R⁷³or R⁷⁴ group is selected from a carboxy, a formyl and a hydroxy, and isattached at the 2-position of the phenyl ring. In some embodiments, inthe structure of Formula XXII at least one R⁷³ or R⁷⁴ group is selectedfrom a carboxy, a formyl and a hydroxy, and is attached at the3-position of the phenyl ring. In some embodiments, in the structure ofFormula XXII at least one R⁷³ or R⁷⁴ group is selected from a carboxy, aformyl and a hydroxy and is attached at the 4-position of the phenylring.

In some embodiments, a compound of the invention is of the structure ofFormula XXIII:

where R⁸¹ is selected from an alkyl, an aryl, an alkoxy, an aryloxy, anacetyl, a carboxy, a formyl, an amido, a hydroxyl, a heterocyclic group,a halo, a nitro and a cyano; and

R⁸² and R⁸³ d independently one or more groups, each R⁸² and R⁸³independently selected from an alkyl, an aryl, an alkoxy, an aryloxy, anacetyl, a carboxy, a formyl, an amido, a hydroxyl, a heterocyclic group,a halo, a nitro and a cyano, where optionally two or more R⁸² and R⁸³groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofFormula XXIV:

where R⁸⁴ and R⁸⁵ are independently selected from an alkyl, an aryl, analkoxy, an aryloxy, an acetyl, a carboxy, a formyl, an amido, ahydroxyl, a heterocyclic group, a halo, a nitro and a cyano; and

R⁸⁶ is one or more groups, each R⁸⁶ independently selected from analkyl, an aryl, an alkoxy, an aryloxy, an acetyl, a carboxy, a formyl,an amido, a hydroxyl, a heterocyclic group, a halo, a nitro and a cyano,where optionally two or more R⁸⁶ groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofFormula XXV:

where R⁸⁷, R⁸⁸, R⁸⁹, R⁹⁰ and R⁹¹ are independently selected from analkyl, an aryl, an alkoxy, an aryloxy, an acetyl, a carboxy, a formyl,an amido, a hydroxyl, a heterocyclic group, a halo, a nitro and a cyano.

In some embodiments, a compound of the invention is of the structure ofFormula XXVI:

where R⁹², R⁹³, R⁹⁴ and R⁹⁵ are independently selected from an alkyl, anaryl, an alkoxy, an aryloxy, an acetyl, a carboxy, a formyl, an amido, ahydroxyl, a heterocyclic group, a halo, a nitro and a cyano; and

R⁹⁶ is one or more groups, each R⁹⁶ independently selected from analkyl, an aryl, an alkoxy, an aryloxy, an acetyl, a carboxy, a formyl,an amido, a hydroxyl, a heterocyclic group, a halo, a nitro and a cyano,where optionally two or more R⁹⁶ groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofFormula XXVII:

where R¹⁰⁰ is selected from an alkyl, an aryl, an acetyl, a sulfonyl anda heterocyclic group;

R¹⁰¹, R¹⁰² and R¹⁰³ are independently selected from an alkyl, an aryl,an alkoxy, an aryloxy, an acetyl, a carboxy, a formyl, an amido, ahydroxyl, a heterocyclic group, a halo, a nitro and a cyano; and

R¹⁰⁵ is one or more groups, each R¹⁰⁵ independently selected from analkyl, an aryl, an alkoxy, an aryloxy, an acetyl, a carboxy, a formyl,an amido, a hydroxyl, a heterocyclic group, a halo, a nitro and a cyano,where optionally two or more R¹⁰⁵ groups may be cyclically linked.

In some embodiments, a compound of the invention is of the structure ofFormula XXVIII:

where R¹⁰⁶ and R¹⁰⁷ are independently one or more groups, each R¹⁰⁶ andR¹⁰⁷ independently selected from an alkyl, an aryl, an alkoxy, anaryloxy, an acetyl, a carboxy, a formyl, an amido, a hydroxyl, aheterocyclic group, a halo, a nitro and a cyano, where optionally two ormore R¹⁰⁶ or R¹⁰⁷ groups may be cyclically linked.

In some embodiments a compound of the invention is of the structure ofone of compounds 6-32 of Table 3. In some embodiments a compound of theinvention is Ro 41-0960 or 3,5-dinitrocatechol (see Table 1).

TABLE 1 Compound Structures

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32Bifunctional Compounds for Disrupting PPIs

Also provided are heterobifunctional compounds that include arecruitment moiety connected to a targeting moiety via a linker. Therecruitment moiety is a ligand of an abundant serum protein (e.g., a TTRbinding compound of the disclosure, as described above). The targetingmoiety is a ligand for a protein target of interest. In someembodiments, the protein target is involved in a protein proteininteraction (PPI) where disruption of the PPI is desirable. For example,the PPI may be important in the regulation of a biological process thatleads to a particular disease condition, where disrupting the PPI ofinterest may provide a method of inhibiting or treating the diseasecondition.

In some embodiments, the heterobifunctional compound is of the formulaR-L-T, where the recruitment moiety R is a TTR-binding compounddescribed by a structure of Table 1; L is a linker; and T is a targetingmoiety. In some cases, the subject heterobifunctional compound includesone or more, such as two or more, recruitment moieties.

The recruitment moiety is connected to the targeting moiety via alinker, at any convenient point of attachment, which may be readilyselected by one of ordinary skill in the art such that the bindingproperty of the ligand to its cognate protein is not significantlyreduced. Exemplary attachment points and strategies of attachmentinclude those described by Gestwicki et al. (Gestwicki, G. R. Crabtree,and I. A. Graef, Harnessing chaperones to generate small-moleculeinhibitors of amyloid beta aggregation. Science, 2004. 306(5697): p.865-9) used to link Congo Red to a synthetic ligand for FKBP, whichstrategy and methods of chemical modification may be readily modifiedfor use in the subject heterobifunctional compounds. In the TTR bindingcompounds described above, the position at which a linker may beconnected using any convenient chemical modification chemistries isdetermined using any convenient selection method, such as but notlimited to, modeling a X-ray crystal structure of TTR (e.g., aco-crystal structure of TTR with a ligand) to determine the mode ofbinding of the recruitment moiety to TTR and to select one or moreappropriate positions which are not involved in contacts with theprotein (e.g., solvent exposed positions), and which may be readilychemically modified. Further methods include determining whether amodification of interest has an adverse effect of the binding of therecruitment moiety to TTR using an in vitro binding assay. Exemplarymodifications of four compounds of Table 1 (compounds Ro41-0960, 7, 14and 9) which may be used to connect these compounds to linkers in thesubject heterobifunctional compounds are shown below:

Any convenient targeting moiety may be used. The targeting moiety may bea small molecule that targets a therapeutic protein target of interest.For example, the targeting moiety may be any convenient binder to aprotein of a target ligand/receptor pair, such as but not limited to,IL2/IL2Rα, TNFα/TNFR1, VEGF-VEGFR, CCL12-CXCR4, CD4-gp120, c-Met-HGF,and LFA-1-CD54. In some embodiments, the targeting moiety is an IL-2ligand such as the IL-2 antagonist Ro26-4550 (FIG. 10). In someembodiments, the targeting moiety is a small molecule TNF-α inhibitor,such as the compound disclosed by He et al. (Science, 2005, 310,1022-1025) (FIG. 10). Suitable positions of the targeting moietiesdescribed above, to which a linker may be attached are selected usingany convenient method, such as but not limited to, modeling methodsusing a X-ray crystal structure of the target protein (e.g., aco-crystal structure of target protein bound with the ligand) to modelthe mode of binding of the targeting moiety and to select appropriatepositions that are not involved in contacts with the target protein(e.g., solvent exposed positions), and which may be readily chemicallymodified. For example, co-crystal structures of the IL-2 and TNF-alphaligands shown in FIG. 10 are available for use in selecting convenientsites in these targeting moieties for chemical modification andattachment of linkers in preparation of subject heterobifunctionalcompounds. Further methods include determining whether a modification ofinterest has an adverse effect of the binding of the targeting moiety tothe target protein using an in vitro binding assay.

Exemplary modifications of IL-2 and TNF-α targeting moieties of interestthat may be used to attach these targeting moieties to linkers inheterobifunctional compounds are shown below:

In some embodiments the protein target of interest is not FKBP, e.g.,the targeting moiety is not a ligand for FKBP.

As used herein, the term “linker”, “linkage” and “linking group” refersto a linking moiety that connects two groups and has a backbone of 20atoms or less in length. A linker or linkage may be a covalent bond thatconnects two groups or a chain of between 1 and 20 atoms in length, forexample of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbonatoms in length, where the linker may be linear, branched, cyclic or asingle atom. In certain cases, one, two, three, four or five or morecarbon atoms of a linker backbone may be optionally substituted with asulfur, nitrogen or oxygen heteroatom. The bonds between backbone atomsmay be saturated or unsaturated, usually not more than one, two, orthree unsaturated bonds will be present in a linker backbone. The linkermay include one or more substituent groups, for example with an alkyl,aryl or alkenyl group. A linker may include, without limitations,oligo(ethylene glycol); ethers, thioethers, tertiary amines, alkyls,which may be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable.

The linking moiety may be conjugated to the recruitment and targetingmoieties using any convenient functional groups (carboxylic acids,amines, alcohols, carbamates, esters, ethers, thioethers, maleimides,and the like), and linking chemistries. For example, any convenientconjugation chemistry described by G. T. Hermanson (“BioconjugateTechniques”, Academic Press, Second Edition, 2008) may be readilyadapted for use in preparing the subject heterobifunctional compounds.

Exemplary linkers that may be used in connecting the recruitment moietyto the targeting moiety using any convenient chemical modificationmethods are shown below:

Dosage Forms of Compounds of the Present Disclosure

In pharmaceutical dosage forms, the compounds disclosed herein may beadministered in the form of their pharmaceutically acceptable salts, orthey may also be used alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. The followingmethods and excipients are merely exemplary and are in no way limiting.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated include but are not necessarily limited to,enteral, parenteral, or inhalational routes, such as intrapulmonary orintranasal delivery.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intrapulmonary, intramuscular, intratracheal,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The composition can be administeredin a single dose or in multiple doses.

For oral preparations, the subject compounds can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents. If oraladministration is desired, the subject compounds may optionally beprovided in a composition that protects it from the acidic environmentof the stomach. For example, the composition can be formulated in anenteric coating that maintains its integrity in the stomach and releasesthe active compound in the intestine. The composition may also beformulated in combination with an antacid or other such ingredient.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes. Parenteral administration can becarried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations. Where local delivery is desired,administration typically involves administering the composition to adesired target tissue, such a liver, heart, spine, etc. For localdelivery, the administration may be by injection or by placement of thecomposition in the desired tissue or organ by surgery, for example.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

The subject compounds of the invention can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqucous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Furthermore, the subject compounds can be made into suppositories bymixing with a variety of bases such as emulsifying bases orwater-soluble bases. The compounds of the present invention can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Dosages of the Compounds of the Present Disclosure

Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus, for example, oral dosages may be about ten times the injectiondose. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from one to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of subject composition to reduce asymptom in a subject animal.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound (s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

Combination Therapy Using the Compounds of the Invention

For use in the subject methods, the subject compounds may be formulatedwith or otherwise administered in combination with otherpharmaceutically active agents, including other protein stabilizingagents, such as resveratrol, heat shock proteins, protein chaperones,and mimics thereof.

The compounds described above may also be administered in combinationwith other therapies for diseases caused by TTR amyloid fibrils.Therapies for diseases caused by TTR amyloid fibrils include hearttransplant for TTR cardiomyopathy, liver transplant, Tafamidis fortreatment of FAP, and the like. The compound described above may beadministered before, after, or during another therapy for diseasescaused by TTR amyloid fibrils.

The compounds described herein for use in combination therapy with thecompounds of the present invention may be administered by the same routeof administration (e.g. intrapulmonary, oral, enteral, etc.) that thecompounds are administered. In the alternative, the compounds for use incombination therapy with the compounds of the present invention may beadministered by a different route of administration that the compoundsare administered.

Kits

Kits with unit doses of the subject compounds, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

Methods

Also provided herein are methods for screening for compounds thatincrease the stability TTR, thereby preventing it from misfolding andforming amyloid fibrils.

Provided herein are methods for using the disclosed compounds toincrease the stability of TTR thereby preventing it from misfolding andforming TTR amyloid fibrils.

Methods for Screening for Protein Stabilizers

A method for screening for TTR stabilizers is provided. The methodgenerally includes screening a candidate compound for stabilizing TTR bycompetitively binding to the same site as bound by a TTR ligand known tobind and stabilize TTR. The method includes: a) contacting aTTR-fluorescence polarization probe (FP probe) complex with a candidatecompound, wherein FP-probe in the TTR-FP probe complex comprises aligand for TTR and a fluorescent moiety attached to the ligand by alinker, wherein the FP-probe stabilizes TTR, and wherein the TTR-FPprobe complex generates a FP signal; and h) determining the FP signal,wherein a decrease in the FP signal indicates the candidate compoundstabilizes TTR.

The method may further comprise determining that the candidate compoundis bound to TTR. The method may further comprise determining theformation of amyloid fibrils by TTR in the presence of the candidatecompound.

The transthyretin used in the screening methods can be wild typetransthyretin or a mutant transthyretin, such as a naturally occurringmutant transthyretin causally associated with the incidence of atransthyretin amyloid disease such as familial amyloid polyneuropathy orfamilial amyloid cardiomyopathy. Exemplary naturally occurring mutanttransthyretins include, but are not limited to, V1221, V30M, L55P (themutant nomenclature describes the substitution at a recited amino acidposition, relative to the wild type; see, e.g., Saraiva et al. (2001)Hum. Mut. 17:493 503).

As noted above, the FP-probe comprises of a ligand and a fluorescencemoiety. The ligand may bind and stabilize TTR and prevent it fromforming amyloid fibrils. Thus, the FP-probe can also bind and stabilizeTTR. In certain embodiments, ligands that bind to and increase thestability of TTR may be already known or may be identified prior toconducting the screen. Methods for identifying ligands and testing theligand for increasing stability of TTR can be designed by the skilledartisan.

In certain embodiments, the ligand may be a ligand known to bind to TTRand increase its stability thereby decreasing its tendency to formamyloid fibrils. A number small molecules that inhibit TTR amyloidformation by binding to the thyroxine (T4) sites in TTR and kineticallystabilizing its quaternary structure are known. Examples of suchstabilizing ligands include Tafamidis, diflunisal, diclofenac analogue,and Resveratrol.

The ligand may be attached to a fluorescent moiety by a linker. Suchlinkers are well known in the art. Generally, the linkers do notcompletely disrupt the binding of the ligand to the protein. In general,the attachment of a linker to the ligand decreases its binding to theprotein by less than 50%, for example by less than 40%, or less than30%, or less than 20%, or less than 10%, or less than 5%, or less than1% or less.

Examples of linker include Polyethylene glycol (PEG), PEG amide, PEGdiamide,N,N-(1,2-aminoethyl);N,N-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-aminoethyl);N,N-(2-[2-(2-aminoethoxy)-ethoxy]-aminoethyl);N,N-[2-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethylcarbamoyl}-ethyldisulfanyl)-aminoethyl];N,N-(amidoacetamido);N-[(5-{2-[2-(2-aminoethoxy)-ethoxy]-ethylcarbamoyl}-pentyl)-carboxamide];N-({5-[2-(2-aminoethyldisulfanyl)-ethylcarbamoyl]-pentyl})-carboxamide;N,N-[(5-aminopentyl)-thioureidyl];N-({2-[2-(2-aminoethoxy)-ethoxy]-ethyl}-carboxamide); or an alkyllinker, optionally including one or heteroatoms in the linker backbone,e.g., —O(CH₂)_(n)—, where n is 1, 2, 3, 4, 5 or 6. The linker may bechosen based on the functional groups present on the ligand and thefluorescent moiety. Linkers that may be used to attach a ligand to afluorescent moiety can be determined by one of skill in the art.

The fluorescent moiety may be any fluorophore or fluorescent group thatwhen excited by light of suitable wavelength can emit fluorescence withhigh quantum yield. See, for example, J. R. Lakowicz in “Principles ofFluorescence Spectroscopy,” Plenum Press, 1983. Numerous knownfluorophores of a wide variety of structures and characteristics aresuitable for use in the practice of methods disclosed here. Typicalfluorescing compounds, which are suitable for use in the presentinvention, include, for example, rhodamine, substituted rhodamine,fluorescein, fluorescein isothiocyanate, naphthofluorescein,dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, andumbelliferone. Other suitable fluorescent groups for use in the presentinvention include, but are not limited to, those described in U.S. Pat.Nos. 4,255,329, 4,668,640 and 5,315,015.

Candidate compounds that may be used in the method for screening forstabilizers of TTR, include numerous chemical classes, primarily organicmolecules, although including in some instances inorganic molecules,organometallic molecules. Also of interest are small organic molecules,which comprise functional groups necessary for structural interactionwith proteins, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups.

Candidate compounds may be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of small molecule compounds. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

A plurality of assays may be run in parallel with a plurality ofdifferent candidate compounds. More than one concentration of one ormore of the candidate compounds may be tested to obtain a differentialresponse to the various concentrations. As known in the art, determiningthe effective concentration of a candidate compound typically uses arange of concentrations resulting from 1:10, or other log scale,dilutions. The concentrations may be further refined with a secondseries of dilutions, if necessary. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection of the agent or at or below theconcentration of agent that does not give a detectable change in FPsignal.

In general, the method comprises contacting a TTR-FP probe complex witha candidate compound. The method uses the polarization signal of theFP-probe. When the FP-probe is free in solution, the FP signal is low.As the FP-probe binds to TTR, the FP signal increases, the increase inthe FP signal is proportional to the decrease in the rate of tumbling ofa FP-probe upon binding to the protein. Thus, the TTR-FP-probe complexgenerates a FP signal. When a candidate compound binds to TTR anddisplaces the bound FP-probe from the protein-FP probe complex, the FPpolarization signal decreases. A decrease in the FP polarization signalindicates that the compound can bind to TTR and displace the stabilizingFP-probe. Since the compound competitively binds to the protein in thepresence of the stabilizing FP-probe, the compound may be more active instabilizing TTR compared to the ligand in the FP-probe.

In general, a decrease in the FP polarization signal indicates that thecandidate compound can bind to TTR and stabilize TTR. The decrease in FPsignal that identifies the candidate compound as a stabilizer for theprotein may be about 10%, or about 20%, or about 30%, or about 40%, orabout 50%, or more.

As noted above, the method may further comprise determining the bindingof the candidate compound to TTR. Any suitable method may be used.Examples of some methods include, Surface Plasmon Resonance (SPR),Isothermal Titration calorimetry (ITC), and the like.

As noted above, the method may further comprise determining theformation of aggregates and/or fibrils by TTR in the presence of thecandidate compound. Such methods are well known in the art. Exemplarymethods for determining formation of aggregates and/or fibrils by TTRare provided below.

In certain embodiments, a method for screening for a candidate compoundthat binds TTR protein is provided. The method includes: a) contacting aTTR-fluorescence polarization probe (FP probe) complex with a candidatecompound, wherein FP-probe in the TTR-FP probe complex comprises aligand for TTR and a fluorescent moiety attached to the ligand by alinker, wherein the FP-probe stabilizes TTR, and wherein the TTR-FPprobe complex generates a FP signal; and b) determining the FP signal,wherein a decrease in the FP signal indicates the candidate compoundbinds TTR.

In general, the compounds disclosed herein as well as the TTRstabilizers identified by the screening methods disclosed above may beused to prevent or reduce the dissociation of a TTR tetramer.

Use of TTR Stabilizers to Decrease TTR Amyloid Formation

The TTR stabilizers disclosed herein and those identified by thescreening methods disclosed above may be used to decrease TTR amyloidformation and to decrease cell dysfunction and/or death associated withTTR amyloid formation. The TTR stabilzers may be used to decrease TTRamyloid formation in vitro in a cell-free system, in vitro in cells, andin vivo.

Amyloid fibril formation may be determined using a turbidity assay invitro in a cell-free system. The turbidity assay can use a wild-type TTRor a mutant of TTR with an increased tendency to form amyloid fibrils.When a wild-type TTR is used TTR amyloidogenesis may be initiated byacidification of TTR. When a mutant of TTR with an increased tendency toform amyloid fibrils, acidification of TTR may also be used.

TTR stabilizers disclosed herein and those identified by the screeningmethods disclosed above may be used to decrease TTR amyloid formation ina cell.

Also provided ides for methods for the stabilization of transthyretin ina tissue or in a biological fluid, and thereby inhibiting misfolding.Generally, the method comprises administering to the tissue orbiological fluid a composition comprising a stabilizing amount of acompound described herein that binds to transthyretin and preventsdissociation of the transthyretin tetramer by kinetic stabilization ofthe native state of the transthyretin tetramer.

Thus, methods which stabilize transthyretin in a diseased tissueameliorate misfolding and lessen symptoms of an associated disease and,depending upon the disease, can contribute to cure of the disease. Theinvention contemplates inhibition of transthyretin misfolding in atissue and/or within a cell. The extent of misfolding, and therefore theextent of inhibition achieved by the present methods, can be evaluatedby a variety of methods, such as are described in the Examples.

Accordingly, in another aspect the invention includes a method oftreating a transthyretin amyloid disease, the method comprisingadministering to a subject diagnosed as having a transthyretin amyloiddisease a therapeutically effective amount of a compound that stabilizesthe native state of the transthyretin tetramer.

In one embodiment, the invention features a method of treating atransthyretin amyloid disease, the method comprising administering to asubject diagnosed as having a transthyretin amyloid disease atherapeutically effective amount of a compound disclosed above thatstabilizes transthyretin tetramer.

The transthyretin amyloid disease can be, for example, familial amyloidpolyneuropathy, familial amyloid cardiomyopathy, or senile systemicamyloidosis.

The subject treated in the present methods can be a human subject,although it is to be understood that the principles of the inventionindicate that the invention is effective with respect to all mammals.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); Room Temperature, RT,rt, and the like.

Materials and Methods

Reagents and Instruments.

Prealbumin from human plasma (human TTR) was purchased from Sigma.EZ-Link Sulfo-NIIS-LC-Biotin was purchased from Pierce. Diclofenac(sodium salt) was purchased from TCI. All reactions were carried outunder nitrogen atmosphere using dry solvents under anhydrous conditions,unless otherwise noted. The solvents used were ACS grade from Fisher.Reagents were purchased from Aldrich and Acros, and used without furtherpurification. Reactions were monitored by thin-layer chromatography(TLC) carried out on 0.20 mm POLYGRAM® SIL silica gel plates (Art.-Nr.805 023) with fluorescent indicator UV₂₅₄ using UV light as avisualizing agent. Normal phase flash column chromatography was carriedout using Davisil® silica gel (100-200 mesh, Fisher). ¹H NMR spectrawere recorded on INOVA 400 MHz spectrometers and calibrated usingresidual undeuterated solvent as an internal reference. Couplingconstants (J) were expressed in Hertz. The following abbreviations wereused to explain the multiplicities: s=singlet, d=doublet, t=triplet,q=quartet, m=multiplet. High-resolution mass spectra (HRMS) wererecorded on a Micromass LCT Electrospray mass spectrometer performed atthe Mass Spectrometry & Proteomics Facility (Stanford University). HPLCanalysis was performed on Waters Delta 600 HPLC system connected to adiode array detector. The samples were analyzed on Xbridge® C18 5 μmreverse phase column (4.6×150 mm) using a linear gradient betweensolvent A (acetonitrile with 0.05% formic acid) and solvent B (waterwith 0.05% formic acid) from 0% to 100% over 10 or 20 minutes at a flowrate of 0.5 ml/min and detected at 254 nm.

Chemical Synthesis

N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2′,6′-difluorobiphenyl-4-carboxamide(Compound 2)

2,2′-(ethane-1,2-diylbis(oxy))diethanamine (280 mg, 1.89 mmol) was addedto a cooled (0° C.) solution of carboxylic acid 1 (22 mg, 0.09 mmol) inanhydrous methylene chloride (2 ml). After 10 min, PyBop (59 mg, 0.11mmol) was added and the reaction was warmed to room temperature andstirred for additional 3 hours. Methylene chloride was added and theorganic layer was washed with semi-saturated sodium bicarbonate solutionand water. The organic layer was dried over sodium sulfate, filtered,and the solvent was removed under reduced pressure. Purification byflash column chromatography (silica gel, 5-10% methanol/methylenechloride) gave 2 (26 mg, 76% yield); ¹H NMR (CD₃OD, 400 MHz) δ 7.93 (d,2H, J=8.4 Hz), 7.56 (d, 2H, J=8.4 Hz), 7.47-7.41 (m, 1H), 7.10 (t, 2H,J=8.2 Hz), 3.72-3.61 (m, 8H), 3.18-3.04 (m, 4H); HRMS (ESI⁺) m/z: calcdfor C₁₉H₂₂F₂N₂O_(3+H) ⁺ 365.1677; found 365.1671 (M+H⁺). A schematic ofthe preparation of compound 2 from compound 1 is shown below. a) PyBop,CH₂Cl₂, 0° C. to RT, 3 h.

tert-butyl-2-(3,5-dichloro-4-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13yloxy)phenylamino)benzoate (35)

To a solution of 33 (177 mg, 0.5 mmol), linker 34 (137 mg, 0.55 mmol),and triphenylphosphine (197 mg, 0.75 mmol) in anhydrous THF (6.0 mL) wasadded a solution of 1,1′-(azodicarbonyl)dipiperidine (ADDP) (189 mg,0.75 mmol) in THF (1.0 ml) dropwise. The reaction was stirred at roomtemperature for 4 days and then concentrated and purified by flashcolumn chromatography (silica gel, 5-20% EtOAc/hexanes) to affordedcompound 35 (148 mg, 51% yield); ¹H NMR (CDCl₃, 400 MHz) δ 9.54 (s, 1H),7.91 (dd, 1H, J=2.0 Hz, 9.5 Hz), 7.37-7.32 (m, 1H), 7.20 (dd, 1H, J=1.0Hz, 8.6 Hz), 7.18 (s, 2H), 6.81-6.76 (m, 1H), 5.08-5.0 (m, 1H), 4.19 (t,2H, J=4.8 Hz), 3.91-3.88 (m, 2H), 3.77-3.74 (m, 2H), 3.67-3.65 (m, 2H),3.57 (t, 2H, J=4.8 Hz), 3.34-3.30 (m, 2H), 1.59 (s, 9H), 1.43 (s, 9H);Low resolution mass spectra for C₂₈H₃₈Cl₂N₂O₇ (ESI⁺): m/z: 585.15(M+H⁺),607.17 (M+Na⁺)

2-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-3,5-dichlorophenylamino)benzoicacid (4)

Trifluoroacetic acid (0.5 ml) was added dropwise to a solution of 35 (5mg, 0.008 mmol) in methylene chloride (2.0 ml) at 0° C. The reactionmixture was warmed to room temperature and stirred for additional 4 h.The solvents were removed under reduced pressure to give thetrifluoroacetate salt of compound 4 (4 mg, 90% yield); ¹H NMR (CD₃OD,400 MHz) δ 8.0 (dd, 1H, J=2.0 Hz, 8.0 Hz), 7.44-7.39 (m, 1H), 7.26-7.22(m, 3H), 6.88-6.82 (m, 1H), 5.02-4.94 (m, 1H), 4.19 (t, 2H, J=4.6 Hz),3.91-3.88 (m, 2H), 3.79-3.76 (m, 2H), 3.74-3.71 (m, 4H), 3.12 (t, 2H,J=4.6 Hz); HRMS (ESI⁺) m/z: calcd for C₁₉H₂₂Cl₂N₂O₅+H⁺ 429.0984; found429.0986 (M+H⁺).

2-(3,5-dichloro-4-(2-(2-(2-(3-(3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-yl)thioureido)ethoxy)ethoxy)ethoxy)phenylamino)benzoicacid (5)

N,N-Diisopropylethylamine (10 μL, 0.057 mmol) was added to a solution ofthe compound 4 (1.9 mg, 0.0035 mmol) and fluorescein isothiocyanate(FITC) (1.2 mg, 0.003 mmol) in DMF (1.0 ml) and the reaction mixture wasstirred at room temperature overnight. The solvent was removed underreduced pressure and the residue was subjected to flash columnchromatography (silica gel, 1-20% methanol/methylene chloride) to givethe desired compound 5 (1.5 mg, 52% yield); 1H NMR (CD₃OD, 400 MHz) δ8.14-8.10 (m, 1H), 7.88 (dd, 1H, J=2.0 Hz, 8.0 Hz), 7.80-7.62 (m, 2H),7.34-7.30 (m, 1H), 7.24-7.18 (m, 1H), 7.18-7.12 (m, 3H), 6.86-6.78 (m,1H), 6.72-6.64 (m, 3H), 6.54-6.50 (m, 2H), 4.21-4.04 (m, 4H), 3.92-3.66(m, 8H); HRMS (ESI⁺) m/z: calcd for C₄₀H₃₃Cl₂N₃O₁₀S+H⁺ 818.1342; found818.1328 (M+H⁺).

A schematic for the synthesis of compounds 35, 4, and 5 is shown below.

a) PPh₃, ADDP, THF, RT, 2 days; b) Trifluoroacetic acid, CH₂Cl₂, 0° C.to RT, 4 h; c) FITC, DIPEA, DMF, RT, 16 h.

Isothermal Titration Calorimetry (ITC).

Calorimetric titrations were carried out on a VP-ITC calorimeter(MicroCal, Northhampton, Mass.). A solution of small molecule (25 μM inPBS pH 7.4, 100 mM KCl, 1 mM EDTA, 8% DMSO) was prepared and titratedinto an ITC cell containing 2 μM of TER in an identical buffer. Prior toeach titration, all samples were degassed for 10 minutes. The initialinjection of 3.0 μL was followed by 30 injections of 8.0 μL each (25°C.) to the point that TTR was fully saturated with ligand. Integrationof the thermogram after subtraction of blanks yielded a binding isothermthat was analyzed with MicroCal Origin 5.0 software.

Fluorescence Polarization Binding Assays

Saturation Binding Experiment.

The binding of probe 5 for TTR was evaluated as follows. In a black96-well plate (Costar), solutions (150 μL final volumes) containing 5(100 nM) were incubated with various concentrations of human TTR (5 nMto 10 μM) in assay buffer (PBS pH 7.4, 0.01% Triton-X100, 1% DMSO) atroom temperature. The samples were allowed to equilibrate by agitationfor 20 min at room temperature. Fluorescence polarization (excitation λ485 nm, emission λ 525 nm, Cutoff λ 515 nm) measurements were takenusing SpectraMax M5 Microplate Reader (Molecular Devices). There was nochange in the FP signal of FITC alone upon addition of TTR, whichindicates that FITC has no interaction with TTR. Nonspecific FP,produced by the free fluorescent probe 5 as well as by binding of 5 tothe plate, was equal to 70±10 mP.

Displacement Binding Experiments for Assay Development.

The affinity of test compounds to TTR was determined as follows. In ablack 96-well plate (Costar), probe 5 (100 nM) was incubated with TTR(200 nM) in assay buffer (PBS pH 7.4, 0.01% Triton-X100, 1% DMSO in 150μL final volumes) at room temperature. Compounds (1,2, Thyroxine, ordiclofenac) were added to the wells in serial dilutions (50 μM to 10nM). All compounds appeared to be soluble under the assay conditions.The samples were allowed to equilibrate by agitation for 20 min at roomtemperature and the fluorescence polarization was measured as describedabove. The data were fit to the following equation[y=(A-D)/(1+(x/C)^B)+D] where A=maximum FP signal, B=slope, C=apparentbinding constant (K_(app)), and D=minimum FP signal. The apparentbinding constant was reported as mean for triplicate experiments and thebest data fit was determined by R² value.

High-Throughput Assay Format.

The HTS FP measurements were performed using black 384-well plates (E&KScientific, # EK-31076) on an Analyst GT (Molecular Devices, Inc.).Approximately 120,000 compounds from the Stanford IITBC library werescreened (Diverse compounds form Chemdiv, Chembridge, SPECS. andLOPAC¹²⁸⁰-1280 compounds Library of Pharmacologically Active Compoundsfrom Sigma). HTS source plates containing 10 mM and 1 mM stock solutionin DMSO were thawed and spun down just prior to testing. 10 μL blankcontrol of assay buffer (PBS pH 7.4, 0.01% Triton-X100) was added tocolumn 24 of the 384 well assay plate and 10 μL of 1.5 nM of the probe 5was added to column 23 of the assay plate. 10 μL mixture of probe 5 (1.5nM) and TTR (50 nM) was added to columns 1 to 22 of the assay plate. 50nL of compounds (10 mM and 1 mM stocks) were then added to columns 2 to22 of the assay plate (compounds were screened at two concentration 50μM and 5 μM). The plates were incubated at room temperature for ˜5minutes and the fluorescence polarization was read in the Analyst GT(top read, bottom of well, Ex 485, Em 530, dichrioc 505, 10 second mix).A second read was also performed after ˜2-5 hours. All FP measurementsare expressed in millipolarization (mP) units calculated using theequation mP=1000X[(I_(S)−I_(SB))−(I_(P)−I_(PB))]/[I_(S)−I_(SB))+(I_(P)−I_(PB))], whereI_(S) is the sample parallel emission intensity measurement, I_(P) isthe sample perpendicular emission measurement, and I_(SB) and I_(PB) arethe corresponding measurement for blank assay buffer. A very gooddynamic range (60 m-220 mP) was observed for the assay.

Determination of IC50 Using the FP Assay.

Serial dilutions of test compounds (at least 8-point duplicate) wereadded to a solution of probe 5 and TTR in assay buffer (15 μL finalvolume). The plates were spun at 1.2K rpm and then read on Analyst GT asdescribed above. A second read was performed after ˜3 hours.

Biotinylation of TTR.

To an ice-cold solution of TTR (20 μM) in PBS was added EZ-LinkSulfo-NHS-LC-Biotin at a ratio of ˜2:1 (biotin:TTR) (minimalbiotinylation) and the solution was incubated at room temperature. After1 hour, un-reacted biotin was removed by passing the sample over a fast(FPLC) desalting column (Superdex™ 75) equilibrated with PBS. This stepwas not sufficient to remove all of the unreacted biotin, therefore, thesample was dialyzed twice against PBS for 2 hours at 4° C.

Surface Plasmon Resonance (SPR) Assay for TTR-Ligands Interaction.

SPR measurements were performed at 25° C. using a Biacore T100 (GEHealthcare) instrument equilibrated in 1% DMSO in PBS as a runningbuffer. After pretreatment of the streptavidin-coated chip (Sensor ChopSA, Biacore), ˜4000 RU biotinylated TTR was immobilized on one channelleaving the second flow channel as a blank (streptavidin alone) control.For binding measurements, HTS hits were diluted in running buffer (1%DMSO/PBS) and different dilution series were flowed over the chip at 50μL/min (150 μL/60 s injection, 600 s wash). A zero concentration samplewas used as a negative control. The kinetic data was fitted to aone-to-one binding model using Biacore T100 evaluation software.

Fibril Formation Assay for TTR Amyloidogenesis.

The efficacy of each compound to inhibit TTR amyloidogenesis wasdetermined by monitoring the turbidity of TTR aggregation at acidic pH,as described previously. 5 μL of test compound (1.44 mM in DMSO) wasadded to a 495 μL of WT TTR (7.2 μM solution in 10 mM phosphate, pH 7.0,100 mM KCl, 1 mM EDTA) in disposable cuvette. The sample was incubatedat room temperature for 30 min after which the pH was lowered to 4.4 byaddition of 500 μL of 100 mM acetate buffer (pH 4.2, 100 mM KCl, 1 mMEDTA) (final compound and TTR concentrations were 7.2 μM and 3.6 μM,respectively). The final 1 ml sample mixture was vortexed, thenincubated at 37° C. for 72 hours, after which the sample was vortexedand the optical density was measured at 400 nm on a Beckman DU® 640spectrophotometer. The percentage of fibril formation was determined bynormalizing the optical density in the presence of test compound by thatof TTR incubated with 5 μL of pure DMSO (representing 100% fibrilformation or 0% inhibition). The optical density was also corrected forthe absorption of all test compounds in the absence of TTR. For thekinetic fibril formation assay (FIG. 5B), the final compound and TTRconcentrations were 3.0 μM and 3.6 μM, respectively. The optical density(400 nm) was measured as described above at 0, 24, 48, 72, 96, and 120hours. All assays were performed in triplicate and the average valuesobtained are presented.

Cell-Based Assay.

Cells:

The human neuroblastoma IMR-32 cell line (CCL-127 ATCC) was maintainedin advanced DMEM (Mediatech, Manassas, Va.), supplemented with 5% FBS, 1mM Hepes buffer, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mLstreptomycin. The human cardiac cell line AC16 was maintained inDMEM:F12 (1:1) supplemented with 10% FBS, 1 mM Hepes buffer, 2 mML-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Two to4-day-old cultures (˜70% confluent) were used for the experiments.

Cell Assays:

Recombinant WT TTR purified in the cold in HBSS (Mediatech, Manassas,Va.) was used as a cytotoxic insult to IMR-32 cells and theamyloidogenic V1221 TTR was used as cytotoxic insult to cardiac AC16cells. Candidate compounds (16 mM in DMSO) were diluted 1:1000 with WTTTR or V1221 TTR (16 mM in HBSS, filter sterilized) or with HBSS only.The mixtures were vortexed and incubated for 24 h at 4° C. WT TTR orV1221 TTR (16 mM in HBSS) containing the same amount of DMSO thanTTR/candidate compound mixtures was prepared in parallel and incubatedunder the same conditions.

The neuroblastoma IMR-32 and the cardiac AC16 cells were seeded in blackwall, clear bottom, tissue culture treated 96-well plates (Costar) at adensity of 6,000 cells/well and 250 cells/well, respectively in Opti-memsupplemented with 5% FBS, 1 mM Hepes buffer, 2 mM L-glutamine, 100units/mL penicillin and 100 μg/mL streptomycin, 0.05 mg/mL CaCl2 andincubated overnight at 37° C. The medium was then removed and replacedimmediately by the TTR/compound or HBSS previously diluted 1:1 inOpti-mem supplemented with 0.8 mg/mL BSA, 2 mM Hepes buffer, 4 mML-glutamine, 200 units/mL penicillin, 200 μg/mL streptomycin and 0.1mg/mL CaCl2. The cells were incubated 24 h at 37° C. after which cellviability was measured. When IMR-32 cells where used the 96 well plateswere spun for 30 min at 1,500×g at 4° C. one hour after TTR/compoundshad been seeded to allow the poorly adherent cells to re-attach to thewells.

Cell Viability Assay:

Cell viability of the cells treated with TTR or TTR/candidate compoundwas evaluated by a resazurin reduction assay. Briefly, 10 μL/well ofresazurin (500 mM, PBS) was added to each well and incubated for 2-3 hat 37° C. Viable cells reduce resazurin to the highly fluorescentresorufin dye, which is quantitated in a multiwell plate reader (Ex/Em530/590 nm, Tecan Safire2, Austria). Cell viability was calculated aspercentage of fluorescence relative to cells treated with vehicle only(100% viability) after subtraction of blank fluorescence (wells withoutcells). All experimental conditions were performed at least in sixreplicates. Averages and SEM corresponding to 2 independently performedexperiments were calculated.

Example 1 Design and Synthesis of the TTR FP Probe

The first step in developing a FP assay is the design of afluoresceinated ligand that binds to TTR. Typically, such ligand wouldbe a TTR binder attached to a fluorescent tag through a suitable linker.The linker is usually included to minimize steric clashes of thefluorophore with binding site residues. Binding is measured by anincrease in the FP signal, which is proportional to the decrease in therate of tumbling of a fluorescent ligand upon association withmacromolecules such as proteins.

Initially, the NSAID diflunisal analogue 1 (FIG. 1A) which has highaffinity for TTR (K_(d)=80 nM) and is a potent inhibitor of TTRaggregation was tested as a fluoresceinated ligand. X-raycrystallographic data showed that compound 1 binds to the TTR T4-bindingsite with its carboxylate-substituted hydrophilic ring oriented in theouter binding pocket and exposed to solvent. Therefore, a poly(ethyleneglycol) (PEG) diamine linker was attached to the carboxylic acid ofcompound 1 by amide bond to produce compound 2 (FIG. 1A). The bindingaffinities of 1 and 2 to TTR were evaluated using isothermal titrationcalorimetry (ITC). As reported earlier, compound 1 was found to have astrong binding affinity to TTR (wild type TTR form human plasma)(K_(d)=72.5±4.7 nM, FIG. 1B). However, attaching a linker to thecarboxylic acid of 1 (compound 2) resulted in a drastic loss of bindingaffinity (K_(d)>3.29 μM, FIG. 1C), which suggests a critical role of thecarboxylic acid of 1 in electrostatic interaction with the Lys 15ε-ammonium groups in the outer pocket of TTR. Wiseman et al. reportedthe NSAID diclofenac analogue 3 (FIG. 1A) as potent TTR aggregationinhibitors. The crystal structure of compound 3 bound to TTRdemonstrates that the phenolic hydroxyl between the two chlorine atomspoints directly out of the binding pocket into the solvent. Thus, thisposition would be a potential site for linker attachment. UsingMitsunobu coupling, a PEG linker was attached to the phenol group ofcompound 3 to generate ligand 4 (FIG. 1A). The binding affinity of 4 toTTR was evaluated using ITC, which demonstrated that compound 4 has verygood affinity for TTR (K_(d)=284.9±58.1 nM, FIG. 10). This affinity iscomparable to the affinity of the known TTR binder diclofenac(K_(d)=370.4±145.4 nM, FIG. 1E). Having confirmed that attaching alinker to 3 did not abrogate the binding affinity, compound 4 was thencoupled to Fluorescein isothiocyanate (FITC) to produce the desiredFITC-coupled TTR probe 5 (FIG. 1A). Attaching FITC to 4 resulted in˜3-fold decrease in binding affinity to TTR (K_(d) for 5=819.7±129.7 nM,FIG. 2A). Fortunately, the affinity of 5 for TTR was not so strong thatunreasonably high concentrations of competitive binders were required todisplace it.

Example 2 Evaluation of the Binding of Probe 5 to TTR Using FP

The binding of probe 5 to TTR was evaluated to test its suitability forthe FP assay.

The binding of 5 for TTR was determined using a standard saturationbinding experiment. A fixed concentration of 5 (100 nM) was incubatedwith increasing concentrations of TTR, and the formation of the 5●TTRcomplex was quantitated using the increase in FP signal (excitation λ485 nm, emission λ 525 nm) (FIG. 2b ). As shown in FIG. 2b , at lowerTTR concentrations, a low mP value was obtained; as the concentration ofTTR increased, a greater fraction of fluorescent 5 bound to TTR andpolarization progressively increased to reach saturation. A very gooddynamic range, of approximately 70-330 mP, was measured for the assay.

Example 3 Displacement FP Assay for TTR Ligands

A displacement assay was used to assess the affinity of various knownTTR binders to TTR. TTR (200 nM) was incubated with 100 nM of 5 and gavean assay window that is close to the maximum value (˜300 mP). Increasingconcentrations of compounds 1 or 2 were added to the 5●TTR complex andthe fluorescence polarization was measured at equilibrium as describedin the experimental procedures. As expected, compound 1, which has astrong binding affinity for TTR, gave a dose dependent decrease in theFP signal which confirms its binding to TTR. An equilibrium bindinganalysis of the data for 1 gave an apparent binding constant (K_(app))of 0.231 μM (R²=0.997) (FIG. 2c ). In comparison, compound 2 (which isnot a TTR ligand, based on ITC as described above) did not displaceprobe 5 from its TTR binding site (no decrease in the FP signal, FIG.3A), which confirms 2 as a poor binder for TTR. Other known TTR binderswere also tested to validate that the FP assay results correlate wellwith a relevant biological parameter. Thyroxine (T4) (K_(app)=0.186 μM,R²=0.998) and diclofenac (K_(app)=4.66 μM, R²=0.999), were able todecrease the FP signal in a dose-dependent manner (FIGS. 3B and 3C). Themain goal of the FP assay describe here is HTS and structure-activityrelationship (SAR) development. Determination of K_(app) of a ligand toTTR is quite sufficient for this purpose because a more informative,though cumbersome, ITC assay is available for more detailed ligandbinding analysis. Importantly, these apparent binding constants,determined by FP assay, correlate very well with the data obtained byITC (Table 2).

TABLE 2 Affinity of ligands as determined by ITC and FP. Ligand ITCK_(d) (μM) FP K_(app) (μM) 1 0.0724 0.231 (R² = 0.997) 2 >3.289 >50Thyroxine (T4) 0.0197^(a) 0.186 (R² = 0.998) Diclofenac 0.370  4.66 (R²= 0.999) ^(a)Detemined by [¹²⁵I]thyroxine (T4) displacent assay ref

Example 4 Adaptation of the FP Assay for HTS

The FP assay was then adapted for HTS and used to screen ˜120,000 smallmolecule library for compounds that displaced probe 5 from the T4binding of TTR. The FP assay was performed in 384-well plate using verylow concentration of probe 5 (1.5 nM) and TTR (50 nM) in a 10 pt assayvolume. A detergent (0.01% Triton-X100) was added to the assay buffer toavoid any false positive hits from promiscuous, aggregate-basedinhibitors. The assay demonstrated robust performance, with a very gooddynamic range (˜70-230 mP) and a Z′ factor in the range of 0.57-0.78(FIGS. 4A and 4B). “Hits” were defined as compounds that resulted in atleast 50% decrease in fluorescence polarization and demonstratedrelative fluorescence between 70 and 130%. Many fluorescence quenchersand enhancers having less than 70% and greater than 130% totalfluorescence relative to a control, respectively, were excluded from thehit list. 200 compounds were designated as positive hits (0.167% hitrate). The 200 hits were then evaluated in a dose-response manner andtheir IC₅₀ (compound concentration that resulted in 50% decrease in theFP signal) values were determined. Among the 200 compounds screened, 33compounds showed a dose-response effect on the FP and displayed IC50≤10μM.

The top 33 hits (compounds with lowest FP IC₅₀) were then purchasedagain and their IC₅₀ was determined in a 10-point duplicatedose-response FP assay (Table 3).

TABLE 3 % fibril forma- IC₅₀ Compounds Structures^(a) tion^(b) (μM)^(c)

 0.43 ± 0.61 0.277

 4.83 ± 1.19 0.289

 0.75 ± 1.06 0.296

 0.37 ± 0.53 0.301

 0.00 ± 0.00  0.3291

 0.52 ± 0.73 0.433

 3.87 ± 0.93 0.504

 5.77 ± 3.91 0.586

10.07 ± 0.53 0.653

 1.08 ± 0.59 0.741

 0.32 ± 0.45 0.755

 1.92 ± 1.08 0.815

 3.22 ± 0.34 0.842

 0.00 ± 0.00 1.152

 0.00 ± 0.00 1.306

 1.81 ± 2.56 2.585

 9.60 ± 1.58 2.656

10.44 ± 4.88 3.349

13.91 ± 1.28 3.707

11.56 ± 0.72 3.742

13.90 ± 5.52 3.927

24.36 ± 1.11 4.217

3.17 ± 4.48 4.786

28.77 ± 6.36 4.881

17.57 ± 4.81 4.933

43.25 ± 4.35 5.414

35.32 ± 3.69 5.604

38.38 ± 3.33 5.803

46.24 ± 1.33 8.69 

48.25 ± 0.68 9.764

50.96 ± 1.98 10.039 

49.43 ± 4.26 10.552 

47.39 ± 1.94 10.957  ^(a)Chemical structure of TTR ligands. ^(b)TTRamyloidogenesis inhibition activity (% fibril formation). ^(c)IC₅₀(compound concentration that resulted in 50% decrease in the FP signal).

Surface plasmon resonance (SPR) was used as an orthogonal biophysicaltechnique to complement the screening assay and further test the hits.Solutions of the 33 hits were passed over immobilized TTR onstreptavidin chip. All compounds identified by the screen as hits wereconfirmed as TTR binders (Based on observed SPR signal). It was shownpreviously that many NSAIDs and isoflavons are potent TTR aggregationinhibitors. Among the potent 33 hits identified in the screen, therewere previously identified NSIADs (diclofenac, meclofenamic acid, andniflumic acid) and isoflavones (apigenin) (Table 3).

Example 5 Evaluation of Amyloidogenesis Inhibition by the HTS HITS

After confirming that the hits hind in the TTR T4-binding site, theefficacy with which these ligands inhibit TTR amyloidogenesis wasevaluated using a previously reported acid-mediated fibril formationassay. This assay has been shown to be equivalent to monitoringamyloidosis using thioflavin T assay. TTR was preincubated with smallmolecules for 30 min at room temperature before amyloidosis wasinitiated (lowering pH to 4.4, incubation at 37° C.). Each compound wasevaluated at a concentration of 7.2 μM relative to a TTR tetramerconcentration of 3.6 μM (equivalent to 7.2 μM because each TTR tetramerhas two T4 sites). This concentration is similar to concentration of TTRin human plasma. The amyloidogenesis inhibition efficacy of eachcompound was determined by monitoring the turbidity of TTR (400 nm). Theextent of TTR amyloidogenesis was quantitated at a fixed time point (72h). All amyloid fibril formation data was normalized to TTRamyloidogenesis in the absence of inhibitor, assigned to be 100% fibrilformation. All the TTR ligands identified in the screen were inhibitorsof TTR aggregation (<50% fibril formation) (Table 3). 23 out of the 33hits were very good aggregation inhibitors (<20% fibril formation whichcorresponds to >80% inhibition of WT TTR fibril formation) among which11 compounds demonstrated excellent TTR aggregation inhibition (<2%fibril formation) (FIG. 5A). Among the potent aggregation inhibitorswere the NSAID niflumic acid and two compounds (3,5-dinitrocatechol andRo 41-0960), which have been reported earlier as selective inhibitors ofcatechol O-methyl transferase (COMT). All other ligands have no reportedbiological activity against TTR amyloidogenesis. The ability of some ofthe potent ligands to inhibit TTR amyloidogenesis at substoichiometricconcentration (TTR 3.6 μM and ligands 3.0 μM) were also tested. Theaggregation inhibition was monitored over 5 days using the acid-mediatedfibril formation assay (compared to 3 days for the fixed time pointdescribed above) (FIG. 5B). Compounds 7, 14, 15, and Ro 41-0960inhibited fibril formation by >75% compared to diclofenac which resultedin only ˜55% inhibition. This clearly indicates that these highly potentligands are able to stabilize the TTR tetramer by binding to only one ofthe two T4 available. The most potent 11 aggregation inhibitors (alongwith niflumic acid as a control) were then evaluated for ability toprevent TTR cytotoxicity against neuron and cardiac cells. The chemicalstructures of all the ligands were confirmed by ¹H NMR andhigh-resolution mass spectrometry (HRMS) and the chemical purity was>95%.

Example 6 Kinetic Stabilizers Prevent TTR-Induced Cytotoxicity AgainstNeurons and Cardiac Cells

In tissue culture, amyloidogenic TTR variants are cytotoxic to cellsderived from tissues that are target of amyloid deposition (Reixach,PNAS 2004). The human neuroblastoma cell line IMR-32 was used as a modelfor FAP (Reixach, PNAS 2004) and a human cardiac cell line AC16 was usedas a model for SSA and FAC.

The amyloidogenic cardiac-specific V1221 TTR variant was used as insultfor the cells of cardiac origin (AC16). Interestingly, thenon-amyloidogenic wild type TTR (WT TTR) purified at 4° C. (as opposedto room temperature) is cytotoxic to AC16, IMR-32 and other neuronalcell lines, possibly because of an altered tetramer structure that mayfacilitate amyloidogenesis (Reixach, BBRC 2006, 348:889-97,Lindhagen-Persson et al, Amyloid 2008 15:240). Cold-purified WT TTR wasused as insult in the IMR-32 system. The advantage of usingcold-purified WT TTR instead of an amyloidogenic variant that depositsin peripheral nerve (like V30M TTR) resides in the fact that WT TTR canbe stored at −80° C. for months without changes in its cytotoxicproperties, whereas other more amyloidogenic TTR variants are not stableand must be re-purified by gel filtration every time before use toremove the large soluble aggregates from solution which will render TTRnon-cytotoxic (Reixach PNAS 2004).

Kinetic stabilizers prevented TTR-induced cytotoxicity by suppressingthe release of monomers which result in misfolding and aggregation,whereas structurally related compounds with poor TTR binding capacity donot inhibit cytotoxicity (Reixach, BBRC 2006, 348:889-97,Lindhagen-Persson et al, Amyloid 2008 15:240).

The compounds under study were pre-incubated with WT TTR or V1221 TTRfor 24 h at 4° C. at equimolar concentrations (8 μM) and added to IMR-32cells or AC16 cells, respectively. Cell viability was measured after 24h by a resazurin reduction assay. Metabolically active cells reduce theresazurin redox dye into resorufin, a soluble compound amenable tofluorescence quantification (O'Brien et al Eur J Biochem 267:5421). Thepercentage of viable cells was calculated relative to cells treated withvehicle only (cell culture media and DMSO)—which were assigned to be100% viable. Resveratrol, a compound known to bind and kineticallystabilize native TTR preventing TTR-induced cytotoxicity was used aspositive control.

The results show that none of the compounds was cytotoxic to IMR-32 orto AC16 cells at the concentration tested (8 μM) (FIGS. 6A and 6B). Cellviability results are reported relative to cells treated with vehicleonly (100% cell viability)(ref). Columns represent the means of twoindependently performed experiments (n=12) and the error bars representstandard errors.

WT TTR and V1221 TTR alone reduced cell viability to 54±1% and 49±1% inIMR-32 and AC16 cells, respectively. For each cell culture assay (FIGS.6C and 6D), the final concentration of both TTR and ligands was 8 μM.Cell viability was assessed using the resazurin reduction assay after 24h. Cell viability results are reported relative to cells treated withvehicle only (100% cell viability)(ret). Columns represent the means oftwo independently performed experiments (n=12) and the error barsrepresent standard errors. Most of the compounds prevented TTR-inducedcytotoxicity in both cell lines (FIGS. 6C and 6D). 3,5-dinitrocatecholwas the only compound found to be poor inhibitor in both tissue culturesystems. The NSAID niflumic acid was also a relatively poor inhibitor inthe cardiac tissue culture system. It must be noted, however that eventhese so-called poorer inhibitors are able to prevent TTR-inducedcytotoxicity in some degree in both cell lines (>68% cell viabilitycompared to ˜50% of TTR alone). Interestingly, compounds Ro 41-0960, 7,14, and 15 demonstrated impressive potency and completely preventedTTR-induced cytotoxicity against both cardiomyocytes and neurons (FIGS.6C and 6D).

Example 7 Determination of Binding Constants of HITS to TTR

Thyroxine (T4) and most reported TTR ligands bind TTR with negativecooperativity. The negative cooperativity of T4 binding to the two T4sites appears to arise from conformational changes in TTR upon bindingof T4 to first site. It is established that occupancy of only one T4binding site within TTR is sufficient to impart kinetic stabilization onthe entire tetramer. Thus, inhibitors exhibiting strong negativecooperativity would be ideal TTR stabilizers. ITC was used determine thebinding constants of some of the hits to TTR and to evaluate presence orabsence of cooperativity between the two TTR T4 sites. For most of theligands studied, there are clearly two phases in the binding isotherms:a strong binding of the first ligand that almost saturates before thesecond weaker-affinity interaction becomes visible. This stronglyindicates that these compounds exhibit negative cooperativity in bindingto TTR. However, attempts to fit the data to interacting sites model ofnegatively cooperative binding yielded poor fits. Integration of thethermograms and subtraction of blanks gave binding isotherms that fitbest a model that considers one set of binding sites. Dissection of thefree energies associated with ligands binding to TTR suggests that theseligands interact differently with TTR (4).

TABLE 4 Isothermal Titration Calorimetry (ITC) analysis of the bindingof ligands to human wild type TTR. Compounds K_(d) (nM) ΔG ΔH TΔS 4284.9 ± 58.1 −8.93 ± 0.14 −8.85 ± 0.95 0.08 ± 0.01 5  819.7 ± 129.7−8.30 ± 0.45 −6.86 ± 0.36 1.44 ± 0.07 1 72.5 ± 4.7 −9.74 ± 0.10 −31.46 ±0.37  −21.72 ± 0.26  7 320.5 ± 27.6 −8.86 ± 0.17 −9.01 ± 0.13 −0.15 ±0.002 Ro 41-0960 184.5 ± 26.5 −9.18 ± 0.47 −13.5 ± 0.32 −4.3 ± 0.11 14 245.1 ± 40.1 −9.02 ± 0.19 −6.66 ± 0.22 2.36 ± 0.08 Niflumic acid  591.7± 146.6 −8.49 ± 0.32 −18.1 ± 1.71 −9.57 ± 0.91  Diclofenac  370.4 ±145.4 −8.78 ± 0.59 −4.69 ± 0.42 4.08 ± 0.37 Dissociation Constants(K_(d)) associated with the binding of ligands to TTR are expressed innM. Thermodynamic Binding Parameters associated with ligand binding toTTR; Binding free energies (ΔG), enthalpies (ΔH), and binding entropies(TΔS) values are reported in units of kcal mol⁻¹.Dissociation Constants (K_(d)) associated with the binding of ligands toTTR are expressed in nM. Thermodynamic Binding Parameters associatedwith ligand binding to TTR; Binding free energies (ΔG), enthalpies (ΔH),and binding entropies (TΔS) values are reported in units of kcal mol⁻¹.

Ro 41-0960 exhibits an enthalpically driven (ΔG=−9.18±0.47 kcal mol⁻¹,ΔH=−13.5±0.32 kcal mol⁻¹) binding to TTR with unfavorable entropy(TΔS=−4.3±0.11 kcal mol⁻¹) (FIG. 7A). This mode of binding is similar tothat of known TTR ligands such as niflumic acid (ΔG=−8.49±0.32 kcalΔH=−18.1±1.71 kcal mol⁻¹ and TΔS=−9.57±0.91 kcal mol⁻¹) (FIG. 7B) and 1(ΔG=−9.74±0.10 kcal mol⁻¹, ΔH=−31.46±0.37 kcal mol⁻¹ and TΔS=−21.72±0.26kcal mol⁻¹). The binding of 7 to TTR is primarily enthalpically driven(ΔG=−8.86±0.17 kcal mol⁻¹, ΔH=−9.01±0.13 kcal mol⁻¹) and there is onlymirror effect of the entropy associated with binding (TΔS=−0.15±0.002kcal mol⁻¹) (FIG. 7C). On the other hand, there is strong favorableentropic contribution to the binding of compound 14 to TTR(TΔS=2.36±0.08 kcal mol⁻¹) (ΔG=−9.02±0.19 kcal mol⁻¹, ΔH=−6.66±0.22 kcalmol⁻¹) (FIG. 7D), which is similar to the favorable entropiccontribution to the binding of diclofenac (TΔS=4.08±0.37 kcal mol⁻¹)(ΔG=−8.78±0.59 kcal mol⁻¹, ΔH=−4.69±0.42 kcal mol⁻¹). The binding of 14to TTR does not cause major conformational changes the TTR tetramerstructure. Thus, the favorable entropy associated with binding of 14,which is larger in size than other ligands, to TTR may be attributed toits ability to displace more water for the hydrophobic T4 site, and notdue to ligand-induced conformational changes with the TTR tetramer.

The interaction of some TTR ligands was further studied using SPR. Thepotent TTR ligand 7 exhibited binding to TTR in aconcentration-dependent with a K_(d) of 57.91±13.2 nM, determined byfitting steady state data (FIG. 7E). This difference between the K_(d)values, form ITC and SPR, may be tolerated by considering the accuracyof the curve fitting and the effects of immobilization of the protein inthe SPR experiment. Measurement of the binding kinetics of 7 to TTR gavea similar binding constant (K_(d)=20.22±2.04 nM) to the one determinedfrom the steady state data. The kinetic data also provided us with animportant parameter in the discovery of selective and drug-likemolecules; target residence time (t). Target residence time, thereciprocal of k_(off), is used differentiate between transient andlong-lived ligand-protein complexes. Compounds that dissociate fromtarget slowly would have longer τ, allowing less frequent administrationof higher doses. Compound 7 has favorable binding kinetics to TTR withrapid on-rate and a slow off-rate (longer τ) (k_(on)=1.29×10⁶±1.3×10⁵M⁻¹ s⁻¹ and k_(off)=0.026±0.001 s⁻¹). Ro 41-0960 also showed similarbinding kinetics to those observed for IG3 (K_(d)=56.05±4.14 nM,k_(on)=2.35×10⁶±1.5×10⁵ M⁻¹ s⁻¹ and k_(off)=0.132±0.009 s⁻¹), though thek_(off) was ˜5× faster (FIG. 8A). In contrast, NSAIDs such as niflumicacid and diclofenac displayed a much faster dissociation form TTR(k_(off)=0.523 s⁻¹ and 1.01 s⁻¹, respectively) compared to 7 or Ro41-0960 (˜20× or 40× lower target residence time than 7) (FIGS. 8B and8C).

Example 8 Heterobifunctional Compounds for PPI Disruption

Recruitment Moiety:

To target extracellular PPIs a number of extracellular proteins wereexamined. The abundant serum protein transthyretin (TTR) was selected asa presenter protein for inhibitors of extracellular receptor/ligandinteractions. Transthyretin is a 55-kDa homotetrameric protein composedof four identical (3-sheet sandwich subunits. The TTR homotetramericstructure possesses 2,2,2 symmetry and is arranged as a dimer of dimers.TTR transports thyroxine (T4) and holoretinol binding protein (RBP) inthe blood and in the cerebrospinal fluid (CSF) using non-overlappingbinding sites [1]. The more labile dimer-dimer interface of the TTRtetramer creates two funnel-shaped T4 binding sites that are 99%unoccupied in the blood, because thyroid-binding globulin and albumintransport the vast majority of T4 in human blood [4]. TTR is desirableas an endogenous presenter protein for extracellular targets because:(1) TTR is present at a concentration of approximately 0.2 mg/mL (3.6μM) in human plasma [13]; (2) TTR is an abundant protein incerebrospinal fluid, present at approximately 10-fold lowerconcentration than in plasma [13]; (3) Several structurally diversesmall molecules, including biaryls, flavones, phenoxazines anddiarylamines have been found to bind to the same binding pocket thatbinds to T4 [14]. A fluorescence polarization (UP) assay was used toidentify TTR ligands and to perform structure-activity relationship(SAR) analysis, as described above. The FP assay was then adapted forHTS and used to screen a ˜120,000 member small molecule library forcompounds that displaced the FP probe from the T4 binding of TTR. “Hits”were defined as compounds that induced at least 50% decrease influorescence polarization and demonstrated relative fluorescence between70 and 130%. 200 compounds were designated as positive hits (0.167% hitrate). The 200 hits were then evaluated in a dose-response manner andtheir IC50 (compound concentration that resulted in 50% decrease in theFP signal) values were determined. The top 100 hits were then purchasedand their IC50 was confirmed again in >10-point duplicate dose responseFP assay. 32 compounds displayed an IC50<1 μM and the binding affinitiesof these compounds to TTR were evaluated using ITC and Surface PlasmonResonance (SPR) (FIG. 5). The cytotoxicity of these compounds wasevaluated and found that only one of the compounds was slightly toxic toprimary cardiomyoctes and neurons (FIG. 6). HTS is also performed usinga red-shifted dye (e.g., Cy3B) in labeling the FP TTR ligand instead ofa FITC based ligand, for increased intensity and lifetime of approx. 2.9ns.

Selection of the Linker.

The linker is selected considering the biophysical properties of theinteracting proteins (Rp: recruited protein, and Tp: target protein).Given the bifunctional compound effectively acts to form a proteincomplex, the long-range forces that govern protein-protein interactionare considered when selecting an appropriate linker. The effect of theelectrostatic interaction may be favorable, unfavourable or small (FIG.9A). If the effect is small, the linker is selected to merely be longenough to project the targeting moiety (T) out of the binding pocket. Ifthe interaction between Rp (recruited protein) and Tp (target protein)is disfavoured the linker is selected to be longer. The longer thelinker (e.g., a flexible linker), the greater the area explored by thetargeting element (FIG. 9B). Although this allows the targeting elementto bind to the protein Tp, the effective concentration of the Tdecreases as the area explored increases. The flexibility of the linkeralso introduces an entropic cost for small flexible linkers, due to therestriction of conformational states in the final bifunctional complex.As the linker increases in length this penalty is reduced. Introductionof rigid elements into the linker restricts the conformational spaceexplored by T, and provided it is sufficiently long allows it to projectfrom the protein, with a reduced conformational penalty. Using a seriesof linker systems, a library of bifunctional molecules is prepared for awide variety of interacting proteins.

Linkers of various length, hydrophilicity, and rigidity are used in thepreparation of bifunctional compounds. The linkers are attached toactivated functional groups on the small molecule targeting andrecruitment moieties using any convenient organic coupling reactions(e.g., ester, amide, and ether bond formation reactions). The affinityof the recruitment complex is modeled using a computational tool(Ocker), and recruitment moieties having known crystal structures, wherethe tool overlays the common elements and produces an energeticallyfavorable complex. Favorable positions for linker attachment areselected using the computational tool and crystal structures.

Selection of Targeting Moieties.

Any convenient small molecule binder of a target protein or targetprotein/receptor pair can be used. In addition, targeting moieties areidentified by small molecule microarrays (SMM) screening for binding tothe target protein using computational approaches [15]. The IL-2antagonist Ro26-4550 (IC50 3-6 μM) is a small molecule inhibitor of thetarget IL-2 (FIG. 10) [16]. The structure of IL-2 bound to the IL-2Rα(Kd≈10 nM) has been described [17] (FIG. 10). Analysis of the crystalstructure shows that the IL-2Rα completely envelops the footprintcovered by the small molecule competitive inhibitor. A linker is addedto Ro26-4550 at a position that points away from IL2 (as determined byanalysis of the X-ray crystal structures of IL-2 bound to the IL2Rα,IC50=10 nM, and Ro26-4550 bound to IL2, IC50=6 uM) and towards pointingaway from IL2 and towards the IL2Rα. Ro26-4550 is covalently linked to anumber of TTR ligands (FIG. 10). The attachment site for the linker onthe TTR ligand is determined by the co-crystal structure of the ligandbound to TTR. Inhibition of IL2 to IL2Rα binding is determined usingstandard assays. In addition, the inhibition of IL2R activation istested using cellular assays, such as IL2 dependent cell proliferation.

TNFα/TNFR1 interaction: A TNFα inhibitor that inhibits in vitro TNFreceptor 1 (TNFR1) binding to TNF-α with an IC50 of 22 μM [18] is usedas a targeting moiety in the bifunctional compounds. TNFα to TNFR1binding assays are performed using standard protocols. In addition,simple cellular assays are performed, such as the inhibition of TNF-αmediated stimulation of inhibitor of NF-κB (IκB) degradation in HeLacells compared to the orthogonal interleukin-1β (IL-1β)-mediatedstimulation of the same pathway, to test the potency of the bifunctionalcompounds to inhibit the interaction between TNF-α and TNFR1.

REFERENCES

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for screening a candidate compound fortransthyretin (TTR) stabilizing activity, the method comprising: a)contacting a TTR-fluorescence polarization (FP) probe complex with acandidate compound, wherein the TTR-FP probe complex comprises TTR andan FP probe, the FP probe comprises a ligand for TTR and a fluorescentmoiety attached to the ligand for TTR by a linker, and the TTR-FP probecomplex generates an FP signal; and b) determining the FP signal,wherein a decrease in the FP signal after addition of the candidatecompound indicates the candidate compound stabilizes TTR.
 2. The methodof claim 1, wherein the TTR-FP probe complex is generated by contactinga sample comprising TTR with the FP probe.
 3. The method of claim 1,wherein TTR in the TTR-FP probe complex is wild type TTR, a mutant TTR,or a combination thereof.
 4. The method of claim 3, wherein TTR in theTTR-FP probe complex is wild type TTR.
 5. The method of claim 3, whereinTTR in the TTR-FP probe complex is a mutant TTR.
 6. The method of claim5, wherein the mutant TTR includes at least one mutation selected fromthe group consisting of V1121, V30M, and L55P.
 7. The method of claim 1,wherein the ligand for TTR is selected from the group consisting ofdiflunisal, diclofenac, tafamidis, and resveratrol.
 8. The method ofclaim 1, wherein the ligand for TTR is NASID diclofenac analogue 3:


9. The method of claim 1, wherein the linker is selected from the groupconsisting of polyethylene glycol (PEG), PEG amide, PED diamide,N,N-(1,2-aminoethyl),N,N-(2-{2-[2-(2-aminoethoxy)-ethoxyl]-ethoxy}-aminoethyl),N,N-(2-[2-(2-aminoethoxy)-ethoxy]-aminoethyl),N,N-[2-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethylcarbamoyl}-ethyldisulfanyl)-aminoethyl],N,N-(amidoacetamido),N-[(5-{2-[2-(2-aminoethoxy)-ethoxy]-ethylcarbamoyl}-pentyl)-carboxamide],N-({5-[2-(2-aminoethyldisulfanyl)-ethylcarbamoyl]-pentyl})-carboxamide;N,N-[(5-aminopentyl)-thioureidyl],N-({2-[2-(2-aminoethoxy)-ethoxy]-ethyl}-carboxamide), and an alkyllinker, wherein the alkyl linker optionally includes one or moreheteroatoms in the linker backbone.
 10. The method of claim 1, whereinthe linker is polyethylene glycol (PEG).
 11. The method of claim 1,wherein the fluorescent moiety is selected from the group consisting ofrhodamine, substituted rhodamine, fluorescein, fluoresceinisothiocyanate, naphthofluorescein, dichlorotriazinylamine fluorescein,dansyl chloride, phycoerythrin, and umbelliferon.
 12. The method ofclaim 1, wherein the fluorescent moiety is fluorescein isothiocyanate.13. The method of claim 1, wherein the FP probe is the chemicalstructure shown below.


14. The method of claim 1, wherein the candidate compound is selectedfrom the group consisting of an organic molecule, an inorganic molecule,and an organic molecule.
 15. The method of claim 1, wherein thecandidate compound is an organic molecule comprising an aromatic moiety.16. The method of claim 1, wherein the candidate compound stabilizes TTRwhen there is at least a 10%, 20%, 30%, 40%, or 50% decrease in FPsignal.
 17. The method of claim 1, wherein the candidate compoundstabilizes TTR when there is at least a 50% decrease in FP signal. 18.The method of claim 1, wherein the method for screening a candidatecompound for TTR stabilizing activity is a high throughput screen (HTS)comprising a plurality of candidate compounds tested in parallel.